2.18. IHC data analysis

For quantification of microvessels, collagen and keratin, tissue sections were viewed at x100 magnification (x10 objective lens and x10 ocular lens). Six fields per tumor section were randomly analyzed. The average number of vessels (and keratin/collagen deposits) was counted and expressed as “number/field”. Proliferation index was estimated as average percentage of PCNA positive cells from microscopy analysis of 6 fields per slide. Average staining of IL8, IL6, CXCL2, MMP1 and PTGS2 proteins were quantified by deconvolution of IHC microphotographs using the IHC profiler plug-in of Image J(25) applied in 5 fields/tumor; the most frequent score was selected; IHC score: 3+ (highly positive); 2+ (positive); 1+ low-positive; 0+ negative. With IHC analysis, peripheral surrounding connective and central necrotic areas were excluded. Histologic slides were blind-coded during the assessment.